Project Summary/Abstract With the rapid advances in DNA sequencing, we now have a near-complete human genome, a fairly comprehensive catalog of germline and somatic variants, as well as rich annotations of functional genomic elements. The next challenge in the field is to obtain a complete functional annotation of genetic variants and genomic elements at the cellular and organismal levels. Genome editing technology, in particularly the CRISPR/Cas9 system, has allowed rapid and precise modifications of the genome and connecting of these to functional outcomes. However, all current high-throughput screening approaches rely on phenotypes that can be coupled to cell survival, cell imaging, fluorescent cell sorting, or affinity enrichments. Genetic variants that have more subtle phenotypic consequences, which might represent the majority, are not amenable to such screens. Furthermore, screening of natural genetic variation via assaying of individual cell lines under in vitro culture conditions also has limited throughput and might miss functional differences that depend on specific physiological contexts. In this project we seek to develop a next-generation functional genetic screening method that can overcome these current limitations. Utilizing induced pluripotent stem cell differentiation as an exemplar model system, we will develop and demonstrate our integrated experimental and computational methodology to enable comprehensive and en masse functional screening of coding and non-coding genomic elements. We expect our platform will greatly accelerate the functional annotation of genetic variants, including many variants of unknown significance, across various normal and diseased cell types and tissues.